Sickle cell disease (SCD) is a devastating hereditary disorder that affects more than 13 million people worldwide with health care costs in the U.S. alone exceeding $1 billion per year. The origin of SCD is a mutant hemoglobin molecule (sickle hemoglobin or HbS) that polymerizes into rigid fibers when deoxygenated. These fibers cause large changes in blood rheology and can ultimately result in complete occlusion of blood vessels, tissue infarction, organ damage, and even death. Despite more than 100 years of research, a clear picture of the vaso-occlusive process remains elusive, and the result is a dearth of treatment options and poor quality of life for the millions of individuals who suffer from this disease. Low oxygen concentration is the one necessary condition for morbidity and mortality in sickle disease, but the quantitative relationship between oxygen concentration and sickle cell blood rheology in physiologic regimes of pressure, vessel size, and hemoglobin concentration is unknown. Ultimately, clinical progress in sickle cell disease requires that we understand how low can a patient's tissue oxygen level fall before a vaso-occlusion will occur, how changes in pressure and vessel dilation modulate the probability of vaso-occlusion at different oxygen concentrations, and how sensitive vaso-occlusion is to slight changes in hemoglobin concentration. Thus, our Specific Aims in these studies are to: (1) Determine the quantitative relationship between sickle blood rheology and blood oxygen concentration; (2) Quantify the effect of vessel size and blood pressure on the relationship between sickle blood rheology and oxygen concentration; (3) Quantify the effect of sickle hemoglobin concentration on the relationship between sickle blood rheology and oxygen concentration. Our primary hypothesis is that the relationship between blood viscosity and oxygen concentration comprises multiple functional regimes, characterized by critical oxygen thresholds, and that this relationship is sensitive to vessel size, blood pressure, and HbS concentration in vivo. Because these parameters cannot be controlled in vivo, we will use an in vitro microfluidic platform with oxygen control to systematically vary these parameters and quantify the effects on sickle blood rheology. The phase space of sickle blood rheology, such as the oxygen thresholds, may be modulated by treatments such as drugs that modulate hemoglobin oxygen binding affinity. Thus, the platforms developed here would be ideal for testing such therapies. The parameters uncovered in these studies may also serve as biomarkers for disease severity to indicate which patients are most in need of care or when patients are most at risk of complications. Overall, the results of these studies will be a clearer understanding of how sickle blood rheology depends on critical physiologic parameters, and this work will produce new platforms and biomarkers for patient monitoring and for prioritizing experimental therapies.